Journal: Cell reports

Article Title: mTORC1 regulates the pyrimidine salvage pathway by controlling UCK2 turnover via the CTLH-WDR26 E3 ligase

doi: 10.1016/j.celrep.2024.115179

Figure Lengend Snippet:

Article Snippet: RanBP9 , Cell Signaling Technology , Cat# 14638S; RRID: AB_2798551.

Techniques: Recombinant, Staining, Protease Inhibitor, Plasmid Preparation, Gel Extraction, DNA Purification, Sequencing, Software

Journal: Communications biology

Article Title: Interplay between β-propeller subunits WDR26 and muskelin regulates the CTLH E3 ligase supramolecular complex.

doi: 10.1038/s42003-024-07371-3

Figure Lengend Snippet: Fig. 2 | Muskelin and WDR26 associate with RanBP9 through the same surfaces. A The indicated HA-RanBP9 domain deletion constructs (see supplemental Fig. 2B) were transfected in shRanBP9 HCT116 cells. After 24 h, cells were lysed in whole-cell extract buffer and immunoprecipitated with an anti-HA antibody. Elutions were run on a western blot and analyzed for the presence of the indicated CTLH complex subunits. Experiments were performed in triplicate (n = 3). B The indicated FLAG- muskelin domain deletion constructs (containing a streptavidin binding peptide, see supplementary Fig. 2B) were transfected in muskelin KO HeLa cells. After 24 h, cells were lysed in whole-cell extract buffer and muskelin complexes were isolated with streptavidin beads. Elutions were run on a western blot and analyzed for the presence of the indicated CTLH complex subunits. Experiments were performed in triplicate

Article Snippet: Fourμgof RanBP9 antibody (F-1, sc-271727, Santa Cruz Biotechnology) and mouse IgG (sc-2025, Santa Cruz Biotechnology) were conjugated to 15 μL Dynabeads Protein G (10004D, Invitrogen) for 1 h at 4 °C with end-over-end rotation.

Techniques: Construct, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Isolation

Journal: FEBS letters

Article Title: Skraban-Deardorff intellectual disability syndrome-associated mutations in WDR26 impair CTLH E3 complex assembly.

doi: 10.1002/1873-3468.14866

Figure Lengend Snippet: Fig. 3. WDR26 mutants affect formation of supramolecular WDR26-CTLH E3 assembly. (A) Cartoon of the wildtype supramolecular CTLH E3 assembly and the potential CTLH (sub-) complexes (I, II, and III) caused by WDR26 SKDEAS-associated mutations. Group I: variants maintaining formation of the supramolecular CTLH E3 assembly; group II: variants disrupting CTLH E3 supramolecular assembly but retaining interactions with RANBP9; and group III: variants abolishing both the higher order CTLH E3 assembly and interactions with RANBP9. HA-tagged WDR26 subunits are indicated. (B) Cell lysates of K562 parental, WDR26- and MKLN1-deficient double knockout K562 cells (WDR26/; MKLN1/), and WDR26/; MKLN1/ cells with stably reintroduced HA-tagged WDR26 were fractionated on a continuous 5–40% sucrose gradient, and fractions analyzed by immunoblotting. Fractions with supramolecular assemblies > 670 kDa are indicated with a red box, and smaller subcomplexes with a blue box. (C, D) Cell lysates of K562 WDR26/; MKLN1/ stably reintroduced HA-tagged WDR26 variants from different individuals (Ind#) were fractionated on a continuous 5–40% sucrose gradient, and fractions analyzed by immunoblotting. Supramolecular assemblies and subcomplexes are boxed in red and blue, respectively. Immunoblot data of individual WDR26 variant are grouped into LisH-CTLH-CRA mutants (C) and WDR26 b-propeller mutants (D). Uncropped blots are provided in Figs S1–S3.

Article Snippet: Membranes were incubated over night at 4 °C with the primary antibodies: HA (C29F4) Rabbit mAb (CST, Danvers, MA, USA; 3724S, RRID: AB_1549585, lot 10 + 11, 1 : 2000), RANBP9 Rabbit pAB (Novus Biologicals, Centannial, CO, USA; PAB16671, RRID: AB_10677213, lot 3, 1 : 1000), MAEA Sheep pAb (R&D Systems, Minneapolis, MN, USA; AF7288, RRID: AB_10971438, CGG10119091, 1 : 1000), YPEL5 pAb (Thermo Fisher Scientific; PA5-26957, RRID: AB_2544457, lot VH3047907, 1 : 1000 in 5% BSA, TBS-T), ACTIN (CST; 4967L, RRID: AB_330288, lot 3, 1 : 1000) or DYKDDDDK Tag (D6W5B) Rabbit mAb (CST; 14793S, RRID: AB_2572291, lot 4, 1 : 1000).

Techniques: Double Knockout, Stable Transfection, Western Blot, Variant Assay